Covalent modification of proteins and other biological macromolecules (carbohydrates, proteoglycans, nucleic acids etc.) is widely used in numerous applications of modern-day biotechnology and academic science. The goals typically sought after by such modifications are to extend the chemical, structural and functional repertoire of the biological macromolecule of interest in order to conform the macromolecule to the requirements of a specific application while retaining or extending its inherent functional properties. Examples include covalent incorporation of proteins into polymeric networks for a variety of tissue engineering applications; manipulation of pharmacokinetics and pharmacodynamics of therapeutic proteins by site-specific attachment of polymeric chains (e.g. polyethylene glycol); covalent attachment of proteins to solid support such as surfaces and beads of variable sizes for a plethora of analytical and enrichment applications; covalent attachment of proteins to various drugs, therapeutics, nanoparticles and various nano-scale biosensors in order to improve solubility, biocompatibility, achieve targeting to a specific organ or tissue and influence other pharmacokinetic and pharmacodynamics properties of such entities; covalent crosslinking of proteins to other proteins or non-protein tags (biotin, antibody, DNA or RNA aptamer, receptor or its ligand, chromophores such as GFP and its relatives, dyes: fluorescent or not, fluorescence quenchers, quantum dots, and numerous others) in order to create chimeric macromolecules combining two or more otherwise (biologically) distinct functions in one molecule (e.g. specific enzymatic activity or fluorescence and a high affinity and specificity towards a certain analyte: used as chromogenic and fluorescent detectors in numerous ELISA and array-based applications).
Due to limited stability and solubility in organic solvents, biomacromolecules such as proteins typically have to be dissolved in water-based media in order to be covalently modified with reasonable efficiency. In proteins, naturally occurring chemical moieties capable of participating in non-enzymatic covalent crosslinking under aqueous environment are limited to a handful of ionizable residues, including amine-containing groups (unmodified amino-termini; side chains of lysines, arginines and histidines), thiols (side chains of cysteines), carboxylates (side chains of aspartates and glutamates) and phenolic side chains of tyrosines. Certain post-translational modifications occurring naturally in some proteins offer additional chemistries for covalent modifications, for example, glycoside residues in glycosylated proteins can be oxidized to form highly reactive aldehyde moieties.
Chemical transformations of the said residues in proteins typically involve nucleophile-mediated mechanisms (see, for example, Hermanson, G. T. Bioconjugate Techniques. 2nd Edition. Elsevier Inc. 2008. ISBN: 978-0-12-370501-3), which imposes certain limitations on the types of chemistry that can be performed on a protein of interest. For example, the ionization state of the chemical group (e.g. amine) is coupled to its strength as a nucleophile, with protonated, positively charged amine being less reactive than non-protonated form. Such protonation state of a protein residue of interest can be controlled by maintaining appropriate pH in aqueous environment, for example by incorporation of appropriate buffer. However, because proteins are polyelectrolytes, certain physical properties of proteins, such as solubility, are also pH-dependent and, to a first approximation, are a function of the amino acid sequence of the specific protein. Therefore, certain proteins are unreactive under the pH optimal for their solubility, and vice versa. One example of such behavior is exhibited by Type I collagen, which in its native form is soluble only under a low pH that renders lysine side chains essentially unreactive.
Covalent attachment to the proteins of hydrophilic polymers such as poly(ethylene glycol) (PEG) (PEGylation) is widely used in biotechnological and biomedical applications as a way to increase solubility, to modulate pharmacokinetic and pharmacodynamics properties and to introduce additional, non-natural chemistries for further modifications. Typically this is accomplished through amine-reactive PEGylations. For example, see Francesco M Veronese (2001) Peptide and protein PEGylation: a review of problems and solutions, Biomaterials Volume 22, Issue 5, 1 Mar. 2001, Pages 405-417.
In another example, in U.S. Pat. No. 7,842,667 and US2010/0137510 A1 Seliktar and colleagues teach of attaching acrylated PEG moieties to collagen and fibrinogen in order to generate water-soluble, biodegradable telechelic macromers that can undergo subsequent chain-growth photopolymerization to yield biocompatible scaffolds for tissue engineering. This was achieved by subjecting denatured proteins to Michael-type addition reactions between acrylate moieties attached to PEG and thiol moieties contained by the proteins. The thiols were introduced into fibrinogen by reducing disulfide bridges that were naturally occurring in this protein; the collagen, naturally devoid of cysteine residues, was treated with a thiolating agent under highly denaturing conditions prior to the Michael addition. While PEGylated proteins obtained in such way were indeed more soluble than their precursors, the chemistries involved significantly limit the range of potential applications. For example, Michael addition used to obtain telechelic derivatives can be slow and require control over the pH of the media, which leads to increase in production costs. In turn, acrylate residues introduced by PEGylation with PEG-diacrylate (or its branched, multi-arm versions) undergo subsequent polymerization via a chain-growth mechanism, resulting in products with (potentially) undesirable pharmacokinetic properties (kinetic chains can easily exceed renal exclusion limit). The ability of acrylates to undergo homopolymerization also prohibits the use of a faster, pH-independent radical chemistry for the protein modification in this case.
Therefore, there is a need for simple, efficient, selective, and broadly applicable methods for the covalent modification of proteins.
Thiol-ene reactions are photochemically initiated, free-radical processes that take place between thiols and olefins (enes) via a sequential propagation/chain-transfer processes. Thiol-ene reactions have a number of significant and unique advantages that make them particularly beneficial. These benefits include the ability to photoinitiate the sample without any need for a distinct and possibly cytotoxic initiator species, the ability to process extremely thick (more than 30 cm) samples because of a self-eliminating light intensity gradient, the very low radical concentration present during reaction producing little protein damage from the free radicals, the lack of oxygen inhibition and the ease with which reactants of significantly varying chemistry can be covalently crosslinked. In addition, by virtue of their radical mechanisms, thiol-ene reactions are practically insensitive to the pH of the solution, which makes them ideally suited for protein modifications in a very broad sense: any protein containing appropriate reactive moieties (e.g. thiols, enes) can be covalently modified at any suitable moiety.
Photo-initiated thiol-ene chemistries have been used with limited success in a number of cases for covalent protein modification. Typically, a thiol-containing protein is subjected to a photo-initiated thiol-ene reaction with an ene-containing moiety, such as a synthetic sugar or polymer (PEG) containing an ene moiety (Conte M. L. et al. (2011). Chem. Commun. 47, pp. 11086-11088; Dondoni A. et al. (2009). Chem. Eur. J. 15, pp. 11444-11449). Alternatively, an ene functionality for subsequent thiol-ene reaction can be introduced into the protein by recombinant techniques, such as codon reprogramming (Floyd N. et al. (2009). Angew. Chem. Int. Ed. 48, pp. 7798-7802) or direct chemical modification of a reduced cysteine moiety (Chalker J. M. et al. (2009) Chem. Commun., pp. 3714-3716). The approaches described in the aforementioned examples have a number of significant limitations. First, codon reprogramming techniques typically are applicable to only small, well-soluble proteins to furnish one or two modifications per protein because recombinant proteins with higher number of unnatural residues are typically not expressed at high enough level (Li Y. et al. (2012) Chem. Sci. 3, pp 2766-2770). A larger number of reactive moieties are often needed to be introduced into proteins of variable, often large size, for a variety of biomedical applications, such as generation of cellular scaffolds for tissue engineering. Second, due to the chemical nature of the ene moieties involved, the above photo-induced thiol-ene reactions are relatively slow and require elevated concentrations of initiator and prolonged exposure of proteins to the UV light which often leads to unwanted side reactions, such as thiol-ene coupling to cystine residues (S—S bonds) that are important for structural integrity of certain proteins (Conte M. L. et al. (2011). Chem. Commun. 47, pp. 11086-11088; Dondoni A. et al. (2009). Chem. Eur. J. 15, pp. 11444-11449). Third, the long exposure can lead to free-radical induced damage of the protein. For example, in Li et al. (2012) the reaction time was up to 2 hours and resulted only in 50% yield of conjugated protein. Dondoni et al. (2009) were able to decrease reaction time to 5 minutes by elevating concentration of radical species in the course of transformation, however this resulted in unforeseen modification of cystine residues due to side reactions. Acrylate and methacrylate moieties, such as the ones employed in the applications U.S. Pat. No. 7,842,667 and US2010/0137510 A1, can undergo photo-initiated thiol-ene coupling more readily and thus alleviate some of the aforementioned limitations, but these electron-poor ene moieties tend to readily self-react (homo-polymerize) in parallel with thiol-ene process (Cramer N. B. et al. (2003) Macromolecules 36, pp. 7964-7979) which significantly limits their chemical orthogonality.
It would be desirable to have methods and compositions that are rapid, pH independent, do not require harsh conditions, and where the functional groups do not self-react.